Broad spectrum ErBB ligand binding molecules and methods for preparing and using them

ABSTRACT

Chimeric ErbB ligand binding molecules having detectable binding activity for more ErbB ligands than any one of native ErbB1, ErbB3 or ErbB4 are disclosed. Preferably, the binding molecules bind a broad spectrum and, more preferably, the full spectrum of ErbB ligands. The chimeric ErbB ligand binding molecules generally have a subunit LI derived from one of Erb-B1, 3, or 4 and a subunit LII derived from another distinct ErbB receptor type. The sub-domain, SI, which joins LI and LII can be from either one of the receptor types or can have portions from both. Pharmaceutical compositions that contain the molecules and methods for the treatment of ErbB sensitive diseases are also disclosed.

BACKGROUND

Receptor tyrosine kinases are involved in stimulating the growth of manycancers. In general, receptor tyrosine kinases are glycoproteins whichconsist of (1) an extracellular domain that is able to bind with aspecific ligand, (2) a transmembrane region, (3) a juxtamembrane domainwhich may regulate the receptor activity by, for instance, proteinphosphorylation, (4) a tyrosine kinase domain that is the enzymaticcomponent of the receptor, and (5) a carboxyterminal tail. The ErbBfamily of type 1 receptor tyrosine kinases constitute one importantclass of receptors because of their importance in mediating cell growth,differentiation and survival in many solid tumors. Members of thisreceptor family include ErbB1 (also known as HER1), ErbB2 (HER2/neu),ErbB3 (HER3), and ErbB4 (HER4). More than a dozen ligands interact withthe ErbB-family receptors. For example, EGF, Transforming Growth Factorα (TGFα), and amphiregulin all bind to ErbB1. Isoforms of neuregulin,also known as Heregulin and Neu Differentiation Factor (NDF) havespecific affinity for ErbB3 and ErbB4. Ligands such as betacellulin,heparin-binding EGF and epiregulin bind to both ErbB1 and ErbB4.

It is becoming clear that over expression of ErbB activating ligands cancause uncontrolled cellular proliferation similar to that of aderegulated receptor. In such cases, interference with the binding ofthe activating ligand to its receptor may provide an effectivetherapeutic strategy or that could accentuate current receptor based orother therapies. Therapeutics that interfere with ligand binding toErbB3 may be particularly effective. ErbB3 differs from the otherreceptors in the EGFR family because its tyrosine kinase domain isfunctionally inactive; however, ErbB2/ErbB3 heterodimers transmit themost potent mitogenic signals of any homo- or heterodimer combination ofthe ErbB family. Therefore, ErbB3 is an important target, yet one thatcannot be inhibited through small molecules that target the kinaseregion. Since ErbB3 requires an activating ligand, such as heregulin orNDF, before activated heterodimers can form, molecules that caninterfere with the binding of ErbB3 receptor ligands could be used toblock or interfere with the formation of ErbB dimers and heterodimers.One example of such a molecule would be a molecule that has ligandbinding affinity and can therefore “trap” ligands and effectively reducetheir concentration so that they cannot activate the ErbB3 receptor. Inaddition to ErbB3 ligands, the other known ErbB receptor ligands, havesimilar effects to varying degrees. Thus, binding molecules that cantrap and sequester the full spectrum of ErbB ligands may be of even moreuse in the treatment of cancer.

Several therapeutics exist that have attempted this trapping or “decoy”strategy. For example, Enbrel™ (etanercept-Amgen) is a soluble, modifiedversion of the TNFR receptor that binds and traps the pro-inflammatoryligand TNFα. In addition, a soluble fusion protein of the VEGFR1 andVEGFR2 receptors, called a VEGF Trap, is currently in clinical trialsfor the treatment of both macular degeneration and several forms ofcancer (Regeneron Pharmaceuticals). An ErbB3 trap has also shown potencyin vitro at enhancing the effects of a dual EGFR/ErbB2 inhibitor andreversed GW2974 (a small molecule inhibitor of ErbB1 and ErbB2)resistance in cells treated with NDF.

All currently approved ErbB inhibitors target either EGFR, ErbB2, ErbB3,ErbB4 or combinations of all 4 receptors. However, no therapeutic isknown that interferes with the binding of ligands to multiple ErbBreceptors simultaneously. Clearly, new binding molecules are needed thatcan be used to sequester receptor ligands, such as ErbB ligands, andthereby block ligand binding to multiple ErbB receptors and subsequentreceptor activation. Binding molecules capable of binding all known ErbBligands would be particularly useful.

A number of binding studies have been carried out to determine regionsof ErbB3 that are important to the binding of its ligand, heregulin.Singer, et. al. (2001), J. Biol. Chem. 276, 44266-44274. Other studiesusing chimeric receptors have identified the relative contributions ofthe extracellular domains of ErbB1 and ErbB4 to ligand-specificsignaling. Kim, et. al. (2002), Eur. J. Biochem. 269, 2323-2329. Thesestudies reveal that neuregulin binding to ErbB4 depends much more ondomain I than on domain III and that domain III of ErbB1 is primarilyimportant for EGF binding. However, these studies were conducted on fulllength receptors which span the entire length of the receptors includingthe transmembrane and cytoplasmic domains. These large molecules presentmanufacturing and administration problems potentially leading to lowertherapeutic efficacy.

SUMMARY

Chimeric ErbB ligand binding molecules having detectable bindingactivity for more ErbB ligands than any one of native ErbB1, ErbB3 orErbB4 are disclosed. Preferably, the binding molecules bind a broadspectrum and, more preferably, the full spectrum of ErbB ligands. Thechimeric ErbB ligand binding molecule generally has a subdomain LIderived from one of ErbB3 or 4 and a subdomain LII derived from anotherdistinct ErbB receptor type, such as ErbB1, which are linked. Thesub-domain, SI, can be used to join LI and LII. Subdomain SI can be fromeither one of the receptor types or can have portions from both.

In one embodiment the chimeric ErbB ligand binding molecule can includeat least a portion of LI from ErbB4 linked to at least a portion of LIIderived from ErbB1. LII can be linked to module 1 of SII from ErbB1. AnSI subdomain can be used to link LI and LII subdomains and can bederived from either the ErbB1 or ErbB4 receptor sequences or can be amixture of both. Optionally, the ErbB chimera can be fused to anaggregant such as IgG2Fc.

In one embodiment the chimeric ErbB ligand binding molecule can includeat least a portion of LI from ErbB4; an SI region having a portionderived from ErbB4 and a portion derived from ErbB1, wherein the ErbB4portion switches to the ErbB1 portion in a region having homologybetween the two sequences; at least a portion of LII can be derived fromErbB1 and module 1 of SII of ErbB1.

In one embodiment the chimeric ErbB ligand binding molecule can includeat least a portion of a subunit LI from ErbB3 linked to at least aportion of an LII derived from ErbB1 and module 1 of SII from ErbB1. Thelinking region can be an SI domain from ErbB1, ErbB3 or their mixtures.

In one embodiment the chimeric ErbB ligand binding molecule can includeat least a portion of a subunit LI from ErbB3, an SI region having aportion is derived from ErbB3 and a portion is derived from ErbB1, atleast a portion of LII derived from ErbB1 and module 1 of SII derivedfrom ErbB1.

In one embodiment the chimeric ErbB ligand binding molecule can includeat least a portion of a subunit LI from ErbB4, an SI region having aportion derived from ErbB4 and a portion is derived from ErbB1, at leasta portion of LII from ErbB1 and module 1 of SII from ErbB1.

In one embodiment the chimeric ErbB ligand binding molecule can includeat least a portion of a subunit LI from ErbB3; an SI region having aportion derived from ErbB3 and a portion derived from ErbB1, wherein theErbB3 portion switches to the ErbB1 portion in a region having homologybetween the two sequences; at least a portion of LII derived from ErbB1and module 1 of SII derived from ErbB1.

DNA sequences that encode the disclosed chimeric ErbB ligand bindingmolecules are also contemplated along with sequences that facilitateexpression and host cells for the maintenance and expression of such DNAsequences.

Pharmaceutical compositions that contain the chimeric ErbB ligandbinding molecules and a pharmaceutically acceptable excipient are alsocontemplated.

The chimeric ErbB ligand binding molecule can be used by immobilizing iton a solid support which can in turn be used for binding ErbB ligandssuch that the ligands can be removed from biological fluids,particularly from patients suffering from diseases associated withover-expression of such ligands. The ErbB ligand depleted biologicalfluids can then be replaced in those patients.

Methods for treating patients having diseases that are associated withoverexpression of ErbB ligands are also contemplated that involveadministering a pharmaceutical composition that contain therapeuticallyeffective amounts of the chimeric ErbB ligand binding molecule in apharmaceutically acceptable excipient.

FIGURES

FIG. 1 illustrates several potential subunit structures of a chimericErbB4-ErbB1 chimera. The highlighted sequence shows one specificembodiment of a chimeric ErbB ligand binding molecule starting with theErbB4 sequence.

FIG. 2 illustrates several potential subunit structures of a chimericErbB3-ErbB1 chimera.

FIG. 3 provides an alignment of ErbB1 (bottom sequence) and ErbB4 (topsequence). The highlighted amino acid sequence shows one specificembodiment of a chimeric ErbB ligand binding molecule starting with theErbB sequence.

DETAILED DESCRIPTION

The complete nucleotide sequences of the ErbB1, ErbB2, ErbB3 and ErbB4are known and can be found in Genbank as accession number NM 005228 forErbB1, accession number NM 004448 for ErbB2, accession number M29366 orNM 001982 for ErbB3, and accession number NM 005235 for ErbB4. The fulllength ectodomain, the extracellular domain, for ErbB receptors containsfour sub-domains, sometimes referred to as L1, CR1, L2 and CR2, where Land CR stand for large and cysteine rich, respectively. The sub-domainsare also sometimes known as domains I-IV or alternatively as LI, SI, LIIand SII as they extend from the amino terminus. Amino acid sequences ofthe receptors have been analyzed and the sequences appear to behomologous and have been aligned. Alignments of the ectodomains ofErbB1, ErbB2, ErbB3 and ErbB4 are provided in US Patent Publication No.2006/0234343 in FIGS. 1A and 1B.

The sub-domains are composed of smaller domains known as subregions. Forexample, the S1 or CR1 sub-domain contains 8 disulfide bonded subregionssometimes known as modules which are numbered 1-8 as they extend fromthe amino to carboxy terminal direction. SII contains seven (7) modulesnumbered 1-7. A large ordered loop has been identified in module 5 thatis thought to project directly away from the ligand binding site. Theamino acid sequence in this loop is highly conserved.

Chimeric ErbB ligand binding molecules are disclosed wherein sub-domainsLI, SI, LII and module 1 of SII from at least two different ErbBreceptors are combined in a single binding molecule. For purposes ofthis disclosure, the phrase “chimeric” with respect to the ErbB ligandbinding molecule is intended to mean a single ErbB ligand bindingmolecule made from and containing portions of more than one ErbBreceptor ectodomain. The phrases “ErbB chimera” and “chimeric ErbBligand binding molecule” are used interchangeably in this applicationand are intended to be synonymous and refer to the protein sequence of amonomer. As can be appreciated in some embodiments the chimera moleculesmay dimerize through formation of disulfide bonds.

Surprisingly, it has been found that portions of the amino acid sequenceof ErbB3 or ErbB4 can be combined with portions of ErbB1 through an SIlinking domain to create a chimeric binding molecule that binds ligandsto both of the combined receptors. Thus, the combination of ErbB1 andErbB4 domains can be used to produce a chimeric binding molecule thathas affinity for both heregulin (ErbB4 specific ligand) and TGFα (ErbB1specific ligand).

The switch in the amino acid sequence from one receptor type to anothercan be at any suitable location that provides for broad spectrum andhigh affinity binding of ErbB ligands. In some embodiments the switchcan occur in the SI sub-domain which links LI and LII sub-domains. Insome embodiments the switch will occur in regions where amino acidsequences are homologous or identical between the receptors beingcombined, such as in module 5 of the SI sub-domain.

For purposes of this disclosure the term “homology” is intended to meana region of amino acid sequence having identical or conservative aminoacid substitutions as that term is generally understood in the art. Forexample with respect to an ErbB4/ErbB1 chimera, as shown in FIG. 3, aswitch from the ErbB4 to the ErbB1 sequence can take place in module 5of the SI sub-domain such that the sequence ending in VYNP from theErbB4 sequence is followed by TTYQ from the ErbB1 sequence, as shown bythe highlighted sequence in FIG. 3.

The sequence can extend as far into the ErbB1 LII domain as is requiredfor ErbB1 ligand binding. The entire ErbB1 LII domain can be includedand a portion or all of the subsequent SII region from ErbB1 can also beincluded. FIG. 3 shows an ectodomain amino acid alignment of the LI, SI,LII and module 1 of the SII domain of ErbB4 and ErbB1, ending at aminoacid 501 of ErbB1.

With reference to FIG. 3, one chimeric ErbB embodiment is shown in whichsubdomain LI is derived from ErbB4; the SI region is derived from aportion of the SI region of ErbB4 and switches to a portion of the SIregion of the ErbB1 in module 5. This sequence continues on to includethe ErbB1 LII subdomain and module 1 of the ErbB1 SII domain. This canbe designated as follows: LI (ErbB4)-SI (ErbB4/ErbB1)-LII (ErbB1)-SIIm1(ErbB1). More specifically: LI (ErbB4 amino acids 1-245)-SI (ErbB1starting with amino acid 249)-LII (ErbB1)-SIIm1 (ErbB1 ending with aminoacid 501 according to the numbering of native ErbB1).

All of the amino acid numbering in this application is intended to beexclusive of the native signal peptide.

In certain embodiments the SI domain can be composed of portions of eachof the two sub-domains, as indicated previously. Further, it is alsopossible to introduce substitutions into the amino acid sequence for avariety of purposes. For example, the DNA sequence for the chimericbinding molecule can be changed to remove cysteines so that theformation of aggregates through cysteine-cysteine bonds can be avoided.Substitutions of amino acids in one subdomain can be used to modifyligand binding affinities. For example, an amino acid from ErbB1 can besubstituted into the ErbB4 LI subdomain to make that domain's sequencemore like that of ErbB1 in order to modify the affinity of the moleculeto ErbB ligands. Similarly, amino acid substitutions from ErbB3 or 4 LIIsubdomains can be included into the ErbB1 subdomain. Such substitutionscan also be made in the SI and SII subdomains. Although any number ofsuch substitutions can be considered substitutions of glutamine fromErbB1 for serine in the ErbB4 portion at position 13, tyrosine forserine at position 42, arginine for tyrosine at position 123 arerepresentative examples. Other examples can be identified by one ofskill in the art simply by comparing sequences. Substitutions that arenot homologous can also be considered. For example, asparagine could besubstituted for serine at position 13 rather than the glutamine found inErbB1 or a residue that has intermediate characteristics of the residuesfound in both receptors may be used.

Any of these chimeric molecules could also be fused to other moleculesor portions thereof including: other chimeric receptors (of any growthfactor receptor family) or to sequences that facilitate purification ofthe product. The DNA sequences can be obtained from commercial sourcesand placed in any suitable expression vector and expressed from suitablehosts of which many are known.

In one embodiment the ErbB chimera can be fused with components thatcause aggregative conjugate formation or extend protein half-life. Forexample, the ErbB chimera can be fused to the constant region ofimmunoglobulin molecule such as the Fc region of IgG. For purposes ofthis disclosure one suitable Fc region is known as IgG2Fc, althoughothers are also known in the art and can be used.

For purposes of this application suitable binding affinities areaffinities that are high enough to bind ErbB ligands in a physiologicalmatrix. Preferably, dissociation constants will be no higher than about10-fold to about 100-fold above the dissociation constants of the nativereceptors. More preferably, the dissociation constants for the ErbBchimera will be within 10-fold of their native receptor counterparts andmore preferably within the same order of magnitude. Most preferably thebinding affinities of the chimeric molecules will not be distinguishablefrom the native counterparts. Any affinity that is sufficient to bindand sequester ErbB ligands to thereby prevent or interfere with ligandbinding and activating ErbB receptors is suitable for use and can finduse in the disclosed methods. Binding affinity, which is a proxy forinhibitor potency, of the binding molecules can be measured usingbiosensor technology or by classic binding assays such as ELISA whichare well known in the art.

DNA that encodes the chimeric ErbB ligand binding molecule sequences isalso contemplated. One of skill can appreciate that the genetic code canbe used to prepare suitable DNA sequences and codon preferences forspecific expression hosts can also be incorporated into such sequences.Also contemplated for use with these sequences are additional DNAsequences that can be used for the expression of these DNA sequences. Avariety of these are known. As is well known in the art such sequencescan also be introduced into host cells for the maintenance of the DNAand for its expression and such hosts that include these DNA sequencesare also contemplated.

Pharmaceutical compositions comprising a disclosed chimeric ErbB ligandbinding molecule are also contemplated. Such compositions comprise atherapeutically effective amount of a chimeric ErbB ligand bindingmolecule, and a pharmaceutically acceptable carrier. The term“pharmaceutically acceptable” means approved by a regulatory agency ofthe Federal or a state government or listed in the U.S. Pharmacopeia orother generally recognized pharmacopeia for use in animals, and moreparticularly, in humans. The term “carrier” refers to a diluent,adjuvant, excipient, or vehicle in which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Suitable pharmaceutical excipients include starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, ethanol and thelike in which the chimeric ErbB ligand binding molecule is soluble andis chemically stable. The composition can also contain wetting oremulsifying agents, or pH buffering agents. These compositions can takethe form of solutions, suspensions, emulsion, tablets, pills, capsules,powders, sustained-release formulations and the like. Pharmaceuticallyacceptable carriers include other ingredients for use in formulationssuch as DPPC, DOPE, DSPC and DOPC. Natural or synthetic surfactants maybe used. PEG may be used (even apart from its use in derivatizing theprotein or analog). Dextrans, such as cyclodextran, may be used.Cellulose and cellulose derivatives may be used. Amino acids may beused, such as use in a buffered formulation. Pharmaceutically acceptablediluents include buffers having various contents (e.g., Tris-HCl,acetate, phosphate), pH and ionic strength; additives such as detergentsand solubilizing agents (e.g., Polysorbate 80), anti-oxidants (e.g.,ascorbic acid, sodium metabisulfite), preservatives (e.g., benzylalcohol) and bulking substances (e.g., lactose, mannitol); incorporationof the material into particulate preparations of polymeric compoundssuch as polylactic acid, polyglycolic acid, etc. or into liposomes.Hyaluronic acid may also be used, and this may have the effect ofpromoting sustained duration in the circulation. Such compositions mayinfluence the physical state, stability, rate of in vivo release, andrate of in vivo clearance of the present proteins and derivatives. See,e.g., Remington's Pharmaceutical Sciences, 18th Ed. (1990, MackPublishing Co., Easton, Pa. 18042) pages 1435-1712 which are hereinincorporated by reference. The compositions may be prepared in liquidform, or may be in dried powder, such as lyophilized form. Implantablesustained release formulations are also contemplated, as are transdermalformulations. Liposome, microcapsule or microsphere, inclusioncomplexes, or other types of carriers are also contemplated.

The amount of the active chimeric binding molecule that will beeffective for its intended therapeutic use can be determined by standardclinical techniques. In addition, in vitro assays may optionally beemployed to help identify optimal dosage ranges. Generally, the dailyregimen should be in the range of 0.1-1000 micrograms of the activeagent (API) kilogram of body weight, preferably 0.1-150 micrograms perkilogram. Effective doses may be extrapolated from dose-response curvesderived from in vitro or suitable animal model test systems. Dosageamount and interval may be adjusted individually to provide plasmalevels of the compounds that are sufficient to maintain therapeuticeffect. In cases of local administration or selective uptake, theeffective local concentration of the compounds may not be related toplasma concentration. The dosage regimen involved in a method fortreatment can be determined by the attending physician, consideringvarious factors which modify the action of drugs, e.g. the age,condition, body weight, sex and diet of the patient, the severity ofdisease, time of administration and other clinical factors.

The amount of compound administered will, of course, be dependent on thesubject being treated, on the subject's weight, the severity of theaffliction, the manner of administration, and the judgment of theprescribing physician. The therapy may be repeated intermittently whilesymptoms are detectable or even when they are not detectable. Thetherapy may be provided alone or in combination with other drugs.

A method for treating a patient in need of treatment is disclosed thatincludes obtaining a chimeric ErbB ligand binding molecule that bindsErbB ligands and interferes with the interaction and effect of ligandson the ErbB receptor system of cancer cells, and administering atherapeutically effective amount of the molecule to a patient.Administration can be by parenteral routes such as i.v. administration,direct injection into a solid tumor such as through a syringe orcatheter or by i.p. injection.

In one method of treatment the chimeric ErbB ligand binding moleculescan be immobilized to a solid support such as an apheresis or biocoresupport by standard methods. When the binding molecule is immobilized toa solid support the serum, blood or other biologically relevant fluid ofa patient can be placed in contact with the solid support in theapheresis column to remove ErbB ligands from the fluid. The serum, bloodor fluid can then be reintroduced into the patient.

The binding molecules can also be used in combination therapies. Thus,the chimeric ErbB ligand binding molecule may be administered incombination with one or more additional compounds or therapies,including chemotherapeutic agents, surgery, catheter devices, andradiation. Combination therapy includes administration of a singlepharmaceutical dosage formulation which contains a chimeric ErbB ligandbinding molecule and one or more additional agents. The chimeric ErbBligand binding molecule and one or more additional agent(s) can beadministered in their own separate pharmaceutical dosage formulations ortogether in the same formulation. For example, a chimeric ErbB ligandbinding molecule and a cytotoxic agent, a chemotherapeutic agent or agrowth inhibitory agent can be administered to the patient together in asingle dosage composition or each agent can be administered in aseparate dosage formulation. More specifically, the chimeric ErbB ligandbinding molecule can be used in combination therapies that includetherapeutic agents such as Lapatinib®, Herceptin®, Erbitux® and thelike. Where separate dosage formulations are used, the chimeric ErbBligand binding molecules and one or more additional agents can beadministered concurrently, or at separately staggered times, i.e.,sequentially. One of skill in the art can appreciate that thecombination must be such that the chimeric ErbB ligand binding moleculedoes not interfere, but rather, accentuates the second therapeutic inthe combination.

The following examples are given by way of illustration only and in noway should be construed as limiting the subject matter of the presentapplication. In the following examples, nucleotide sequences that encodethe disclosed amino acid sequences are also contemplated. Many of thefollowing examples disclose the sequence for IgG2Fc fused to the ErbBchimera. It should be appreciated that use of IgGFc is optional. Inaddition, the conservative replacement of an amino acid with anothersimilar amino acid that does not substantially (about 10-fold) interferewith ligand binding activity is specifically contemplated. Conventionalbinding studies of the purified products can be used to determinewhether substantial differences in binding affinities exist. Many of thestructures described below can be better understood by reference to thediagrams of those structures in FIGS. 1 and 2. All sequences include asignal peptide derived from a mouse antibody heavy chain gene. Allnumbering is exclusive of the signal peptide, and the first amino acidof each ErbB sequence is underlined.

EXAMPLE 1

The present example specifically discloses a chimera containing a signalpeptide (M E W S W V F L F F L S V T T G V H S) joined to the LIsubdomain of the ErbB3 receptor and the SI and LII subdomains of theErbB1 receptor terminating after residue 501 in module 1 of the ErbB1SII sub-domain. The ErbB chimera has the following amino acid sequence.

SEQ. ID. NO. 1 M E W S W V F L F F L S V T T G V H S S E V G NS Q A V C P G T L N G L S V T G D A E N Q Y Q TL Y K L Y E R C E V V M G N L E I V L T G H N AD L S F L Q W I R E V T G Y V L V A M N E F S TL P L P N L R V V R G T Q V Y D G K F A I F V ML N Y N T N S S H A L R Q L R L T Q L T E I L SG G V Y I E K N D K L C H M D T I D W R D I V RD R D A E I V V K D N G R S C P P C D P S C P NG S C W G A G E E N C Q K L T K I I C A Q Q C SG R C R G K S P S D C C H N Q C A A G C T G P RE S D C L V C R K F R D E A T C K D T C P P L ML Y N P T T Y Q M D V N P E G K Y S F G A T C VK K C P R N Y V V T D H G S C V R A C G A D S YE M E E D G V R K C K K C E G P C R K V C N G IG I G E F K D S L S I N A T N I K H F K N C T SI S G D L H I L P V A F R G D S F T H T P P L DP Q E L D I L K T V K E I T G F L L I Q A W P EN R T D L H A F E N L E I I R G R T K Q H G Q FS L A V V S L N I T S L G L R S L K E I S D G DV I I S G N K N L C Y A N T I N W K K L F G T SG Q K T K I I S N R G E N S C K A T G Q V C H AL C S P E G C W G P E P R D C V S V E C P P C PA P P V A G P S V F L F P P K P K D T L M I S RT P E V T C V V V D V S H E D P E V Q F N W Y VD G M E V H N A K T K P R E E Q F N S T F R V VS V L T V V H Q D W L N G K E Y K C K V S N K GL P A P I E K T I S K T K G Q P R E P Q V Y T LP P S R E E M T K N Q V S L T C L V K G F Y P SD I A V E W E S N G Q P E N N Y K T T P P M L DS D G S F F L Y S K L T V D K S R W Q Q G N V FS C S V M H E A L H N H Y T Q K S L S L S P G K Stop

EXAMPLE 2

The present example specifically discloses a chimera containing a signalpeptide (M E W S W V F L F F L S V T T G V H S) joined to LI and SIsubdomains of ErbB3 receptor and the LII subdomain of the ErbB1 receptorterminating after residue 501 in module 1 of the ErbB1 SII sub-domain.The ErbB chimera has the following amino acid sequence:

SEQ. ID. NO. 2 M E W S W V F L F F L S V T T G V H S S E V G NS Q A V C P G T L N G L S V T G D A E N Q Y Q TL Y K L Y E R C E V V M G N L E I V L T G H N AD L S F L Q W I R E V T G Y V L V A M N E F S TL P L P N L R V V R G T Q V Y D G K F A I F V ML N Y N T N S S H A L R Q L R L T Q L T E I L SG G V Y I E K N D K L C H M D T I D W R D I V RD R D A E I V V K D N G R S C P P C H E V C K GR C W G P G S E D C Q T L T K T I C A P Q C N GH C F G P N P N Q C C H D E C A G G C S G P Q DT D C F A C R H F N D S G A C V P R C P Q P L VY N K L T F Q L E P N P H T K Y Q Y G G V C V AS C P H N F V V D Q T S C V R A C P P D K M E VD K N G L K M C E P C G G L C P K V C N G I G IG E F K D S L S I N A T N I K H F K N C T S I SG D L H I L P V A F R G D S F T H T P P L D P QE L D I L K T V K E I T G F L L I Q A W P E N RT D L H A F E N L E I I R G R T K Q H G Q F S LA V V S L N I T S L G L R S L K E I S D G D V II S G N K N L C Y A N T I N W K K L F G T S G QK T K I I S N R G E N S C K A T G Q V C H A L CS P E G C W G P E P R D C V S V E C P P C P A PP V A G P S V F L F P P K P K D T L M I S R T PE V T C V V V D V S H E D P E V Q F N W Y V D GM E V H N A K T K P R E E Q F N S T F R V V S VL T V V H Q D W L N G K E Y K C K V S N K G L PA P I E K T I S K T K G Q P R E P Q V Y T L P PS R E E M T K N Q V S L T C L V K G F Y P S D IA V E W E S N G Q P E N N Y K T T P P M L D S DG S F F L Y S K L T V D K S R W Q Q G N V F S CS V M H E A L H N H Y T Q K S L S L S P G K Stop

EXAMPLE 3

The present example discloses a chimera containing a signal peptide (M EW S W V F L F F L S V T T G V H S) joined to the LI subdomain andmodules 1-2 of the SI subdomain of the ErbB3 receptor and modules 3-8 ofthe SI subdomain, the LII subdomain and module 1 of SII subdomain of theErbB1 receptor fused to IgG2Fc. The ErbB chimera has the following aminoacid sequence:

SEQ. ID. NO. 3 M E W S W V F L F F L S V T T G V H S S E V G NS Q A V C P G T L N G L S V T G D A E N Q Y Q TL Y K L Y E R C E V V M G N L E I V L T G H N AD L S F L Q W I R E V T G Y V L V A M N E F S TL P L P N L R V V R G T Q V Y D G K F A I F V ML N Y N T N S S H A L R Q L R L T Q L T E I L SG G V Y I E K N D K L C H M D T I D W R D I V RD R D A E I V V K D N G R S C P P C H E V C K GR C W G P G S E D C Q T L T K T I C A P Q C N GH C F G P N P N Q C C H N Q C A A G C T G P R ES D C L V C R K F R D E A T C K D T C P P L M LY N P T T Y Q M D V N P E G K Y S F G A T C V KK C P R N Y V V T D H G S C V R A C G A D S Y EM E E D G V R K C K K C E G P C R K V C N G I GI G E F K D S L S I N A T N I K H F K N C T S IS G D L H I L P V A F R G D S F T H T P P L D PQ E L D I L K T V K E I T G F L L I Q A W P E NR T D L H A F E N L E I I R G R T K Q H G Q F SL A V V S L N I T S L G L R S L K E I S D G D VI I S G N K N L C Y A N T I N W K K L F G T S GQ K T K I I S N R G E N S C K A T G Q V C H A LC S P E G C W G P E P R D C V S V E C P P C P AP P V A G P S V F L F P P K P K D T L M I S R TP E V T C V V V D V S H E D P E V Q F N W Y V DG M E V H N A K T K P R E E Q F N S T F R V V SV L T V V H Q D W L N G K E Y K C K V S N K G LP A P I E K T I S K T K G Q P R E P Q V Y T L PP S R E E M T K N Q V S L T C L V K G F Y P S DI A V E W E S N G Q P E N N Y K T T P P M L D SD G S F F L Y S K L T V D K S R W Q Q G N V F SC S V M H E A L H N H Y T Q K S L S L S P G K Stop

EXAMPLE 4

The present example discloses a chimera containing a signal peptide (M EW S W V F L F F L S V T T G V H S) joined to the LI subdomain andmodules 1-3 of the SI subdomain of ErbB3 receptor and modules 4-8 of theSI subdomain, the LII subdomain and module 1 of SII subdomain of theErbB1 receptor fused to IgG2Fc. The ErbB chimera has the following aminoacid sequence:

SEQ. ID. NO. 4 M E W S W V F L F F L S V T T G V H S S E V G NS Q A V C P G T L N G L S V T G D A E N Q Y Q TL Y K L Y E R C E V V M G N L E I V L I G H N AD L S F L Q W I R E V T G Y V L V A M N E F S TL P L P N L R V V R G T Q V Y D G K F A I F V ML N Y N T N S S H A L R Q L R L T Q L T E I L SG G V Y I E K N D K L C H M D T I D W R D I V RD R D A E I V V K D N G R S C P P C H E V C K GR C W G P G S E D C Q T L T K T I C A P Q C N GH C F G P N P N Q C C H D E C A G G C S G P Q DT D C F V C R K F R D E A T C K D T C P P L M LY N P T T Y Q M D V N P E G K Y S F G A T C V KK C P R N Y V V T D H G S C V R A C G A D S Y EM E E D G V R K C K K C E G P C R K V C N G I GI G E F K D S L S I N A T N I K H F K N C T S IS G D L H I L P A F R G D S F T H T P P L D P QE L D I L K T V K E I T G F L L I Q A W P E N RT D L H A F E N L E I I R G R T K Q H G Q F S LA V V S L N I T S L G L R S L K E I S D G D V II S G N K N L C Y A N T I N W K K L F G T S G QK T K I I S N R G E N S C K A T G Q V C H A L CS P E G C W G P E P R D C V S V E C P P C P A PP V A G P S V F L F P P K P K D T L M I S R T PE V T C V V V D V S H E D P E V Q F N W Y V D GM E V H N A K T K P R E E Q F N S T F R V V S VL T V V H Q D W L N G K E Y K C K V S N K G L PA P I E K T I S K T K G Q P R E P Q V Y T L P PS R E E M T K N Q V S L T C L V K G F Y P S D IA V E W E S N G Q P E N N Y K T T P P M L D S DG S F F L Y S K L T V D K S R W Q Q G N V F S CS V M H E A L H N H Y T Q K S L S L S P G K Stop

EXAMPLE 5

The present example specifically discloses a chimera containing a signalpeptide (M E W S W V F L F F L S V T T G V H S) joined to the LIsubdomain and modules 1-4 of the SI subdomain of ErbB3 receptor withmodules 5-8 of the SI subdomain, the LII subdomain and module 1 of SIIsubdomain of the ErbB1 receptor fused to IgG2Fc. The ErbB chimera hasthe following amino acid sequence:

SEQ. ID. NO. 5 M E W S W V F L F F L S V T T G V H S S E V G NS Q A V C P G T L N G L S V T G D A E N Q Y Q TL Y K L Y E R C E V V M G N L E I V L T G H N AD L S F L Q W I R E V T G Y V L V A M N E F S TL P L P N L R V V R G T Q V Y D G K F A I F V ML N Y N T N S S H A L R Q L R L T Q L T E I L SG G V Y I E K N D K L C H M D T I D W R D I V RD R D A E I V V K D N G R S C P P C H E V C K GR C W G P G S E D C Q T L T K T I C A P Q C N GH C F G P N P N Q C C H D E C A G G C S G P Q DT D C F A C R H F N D S G A C V P T C P P L M LY N P T T Y Q M D V N P E G K Y S F G A T C V KK C P R N Y V V T D H G S C V R A C G A D S Y EM E E D G V R K C K K C E G P C R K V C N G I GI G E F K D S L S I N A T N I K H F K N C T S IS G D L H I L P V A F R G D S F T H T P P L D PQ E L D I L K T V K E I T G F L L I Q A W P E NR T D L H A F E N L E I I R G R T K Q H G Q F SL A V V S L N I T S L G L R S L K E I S D G D VI I S G N K N L C Y A N T I N W K K L F G T S GQ K T K I I S N R G E N S C K A T G Q V C H A LC S P E G C W G P E P R D C V S V E C P P C P AP P V A G P S V F L F P P K P K D T L M I S R TP E V T C V V V D V S H E D P E V Q F N W Y V DG M E V H N A K T K P R E E Q F N S T F R V V SV L T V V H Q D W L N G K E Y K C K V S N K G LP A P I E K T I S K T K G Q P R E P Q V Y T L PP S R E E M T K N Q V S L T C L V K G F Y P S DI A V E W E S N G Q P E N N Y K T T P P M L D SD G S F F L Y S K L T V D K S R W Q Q G N V F SC S V M H E A L H N H Y T Q K S L S L S P G K Stop

EXAMPLE 6

The present example specifically discloses a chimera containing a signalpeptide (M E W S W V F L F F L S V T T G V H S) joined to the LIsubdomain and modules 1-5 of the SI subdomain of ErbB3 receptor with themodules 6-8 of the SI subdomain, the LII subdomain and module 1 of theSII subdomain of the ErbB1 receptor fused to IgG2Fc. The ErbB chimerahas the following amino acid sequence:

SEQ. ID. NO. 6 M E W S W V F L F F L S V T T G V H S S E V G NS Q A V C P G T L N G L S V T G D A E N Q Y Q TL Y K L Y E R C E V V M G N L E I V L T G H N AD L S F L Q W I R E V T G Y V L V A M N E F S TL P L P N L R V V R G T Q V Y D G K F A I F V ML N Y N T N S S H A L R Q L R L T Q L T E I L SG G V Y I E K N D K L C H M D T I D W R D I V RD R D A E I V V K D N G R S C P P C H E V C K GR C W G P G S E D C Q T L T K T I C A P Q C N GH C F G P N P N Q C C H D E C A G G C S G P Q DT D C F A C R H F N D S G A C V P R C P Q P L VY N K L T F Q L E P N P H T K Y Q Y G G V C V AK C P R N Y V V T D H G S C V R A C G A D S Y EM E E D G V R K C K K C E G P C R K V C N G I GI G E F K D S L S I N A T N I K H F K N C T S IS G D L H I L P V A F R G D S F T H T P P L D PQ E L D I L K T V K E I T G F L L I Q A W P E NR T D L H A F E N L E I I R G R T K Q H G Q F SL A V V S L N I T S L G L R S L K E I S D G D VI I S G N K N L C Y A N T I N W K K L F G T S GQ K T K I I S N R G E N S C K A T G Q V C H A LC S P E G C W G P E P R D C V S V E C P P C P AP P V A G P S V F L F P P K P K D T L M I S R TP E V T C V V V D V S H E D P E V Q F N W Y V DG M E V H N A K T K P R E E Q F N S T F R V V SV L T V V H Q D W L N G K E Y K C K V S N K G LP A P I E K T I S K T K G Q P R E P Q V Y T L PP S R E E M T K N Q V S L T C L V K G F Y P S DI A V E W E S N G Q P E N N Y K T T P P M L D SD G S F F L Y S K L T V D K S R W Q Q G N V F SC S V M H E A L H N H Y T Q K S L S L S P G K Stop

EXAMPLE 7

The present example describes a chimera containing a signal peptide (M EW S W V F L F F L S V T T G V H S) joined to the LI subdomain andmodules 1-6 of the SI subdomain of the ErbB3 receptor with the modules7-8 of the SI subdomain, the LII subdomain and module 1 of the SIIsubdomain of the ErbB1 receptor fused to IgG2Fc. The ErbB chimera hasthe following amino acid sequence:

SEQ. ID. NO. 7 M E W S W V F L F F L S V T T G V H S S E V G NS Q A V C P G T L N G L S V T G D A E N Q Y Q TL Y K L Y E R C E V V M G N L E I V L T G H N AD L S F L Q W I R E V T G Y V L V A M N E F S TL P L P N L R V V R G T Q V Y D G K F A I F V ML N Y N T N S S H A L R Q L R L T Q L T E I L SG G V Y I E K N D K L C H M D T I D W R D I V RD R D A E I V V K D N G R S C P P C H E V C K GR C W G P G S E D C Q T L T K T I C A P Q C N GH C F G P N P N Q C C H D E C A G G C S G P Q DT D C F A C R H F N D S G A C V P R C P Q P L VY N K L T F Q L E P N P H T K Y Q Y G G V C V AS C P H N F V V D Q T S C V R A C G A D S Y E ME E D G V R K C K K C E G P C R K V C N G I G IG E F K D S L S I N A T N I K H F K N C T S I SG D L H I L P V A F R G D S F T H T P P L D P QE L D I L K T V K E I T G F L L I Q A W P E N RT D L H A F E N L E I I R G R T K Q H G Q F S LA V V S L N I T S L G L R S L K E I S D G D V II S G N K N L C Y A N T I N W K K L F G T S G QK T K I I S N R G E N S C K A T G Q V C H A L CS P E G C W G P E P R D C V S V E C P P C P A PP V A G P S V F L F P P K P K D T L M I S R T PE V T C V V V D V S H E D P E V Q F N W Y V D GM E V H N A K T K P R E E Q F N S T F R V V S VL T V V H Q D W L N G K E Y K C K V S N K G L PA P I E K T I S K T K G Q P R E P Q V Y T L P PS R E E M T K N Q V S L T C L V K G F Y P S D IA V E W E S N G Q P E N N Y K T T P P M L D S DG S F F L Y S K L T V D K S R W Q Q G N V F S CS V M H E A L H N H Y T Q K S L S L S P G K Stop

EXAMPLE 8

The present example discloses a chimera containing a signal peptide (M EW S W V F L F F L S V T T G V H S) joined to the LI subdomain andmodules 1-7 of the SI subdomain of the ErbB3 receptor and module 8 ofthe SI subdomain, the LII subdomain and module 1 of the SII subdomain ofthe ErbB1 receptor fused to IgG2Fc. The ErbB chimera has the followingamino acid sequence:

SEQ. ID. NO. 8 M E W S W V F L F F L S V T T G V H S S E V G NS Q A V C P G T L N G L S V T G D A E N Q Y Q TL Y K L Y E R C E V V M G N L E I V L T G H N AD L S F L Q W I R E V T G Y V L V A M N E F S TL P L P N L R V V R G T Q V Y D G K F A I F V ML N Y N T N S S H A L R Q L R L T Q L T E I L SG G V Y I E K N D K L C H M D T I D W R D I V RD R D A E I V V K D N G R S C P P C H E V C K GR C W G P G S E D C Q T L T K T I C A P Q C N GH C F G P N P N Q C C H D E C A G G C S G P Q DT D C F A C R H F N D S G A C V P R C P Q P L VY N K L T F Q L E P N P H T K Y Q Y G G V C V AS C P H N F V V D Q T S C V R A C P P D K M E VD K N G L K M C E P C E G P C R K V C N G I G IG E F K D S L S I N A T N I K H F K N C T S I SG D L H I L P V A F R G D S F T H T P P L D P QE L D I L K T V K E I T G F L L I Q A W P E N RT D L H A F E N L E I I R G R T K Q H G Q F S LA V V S L N I T S L G L R S L K E I S D G D V II S G N K N L C Y A N T I N W K K L F G T S G QK T K I I S N R G E N S C K A T G Q V C H A L CS P E G C W G P E P R D C V S V E C P P C P A PP V A G P S V F L F P P K P K D T L M I S R T PE V T C V V V D V S H E D P E V Q F N W Y V D GM E V H N A K T K P R E E Q F N S T F R V V S VL T V V H Q D W L N G K E Y K C K V S N K G L PA P I E K T I S K T K G Q P R E P Q V Y T L P PS R E E M T K N Q V S L T C L V K G F Y P S D IA V E W E S N G Q P E N N Y K T T P P M L D S DG S F F L Y S K L T V D K S R W Q Q G N V F S CS V M H E A L H N H Y T Q K S L S L S P G K Stop

EXAMPLE 9

The present example discloses a chimera containing a signal peptide (M EW S W V F L F F L S V T T G V H S) joined to the LI subdomain and module1 of the SI subdomain of the ErbB3 receptor and modules 2-8 of the SIsubdomain, the LII subdomain and module 1 of the SII subdomain of theErbB1 receptor fused to IgG2Fc. The ErbB chimera has the following aminoacid sequence:

SEQ. ID. NO. 9 M E W S W V F L F F L S V T T G V H S S E V G NS Q A V C P G T L N G L S V T G D A E N Q Y Q TL Y K L Y E R C E V V M G N L E I V L T G H N AD L S F L Q W I R E V T G Y V L V A M N E F S TL P L P N L R V V R G T Q V Y D G K F A I F V ML N Y N T N S S H A L R Q L R L T Q L T E I L SG G V Y I E K N D K L C H M D T I D W R D I V RD R D A E I V V K D N G R S C P P C H E V C K GR C W G P G S E D C Q T L T K I I C A Q Q C S GR C R G K S P S D C C H N Q C A A G C T G P R ES D C L V C R K F R D E A T C K D T C P P L M LY N P T T Y Q M D V N P E G K Y S F G A T C V KK C P R N Y V V T D H G S C V R A C G A D S Y EM E E D G V R K C K K C E G P C R K V C N G I GI G E F K D S L S I N A T N I K H F K N C T S IS G D L H I L P V A F R G D S F T H T P P L D PQ E L D I L K T V K E I T G F L L I Q A W P E NR T D L H A F E N L E I I R G R T K Q H G Q F SL A V V S L N I T S L G L R S L K E I S D G D VI I S G N K N L C Y A N T I N W K K L F G T S GQ K T K I I S N R G E N S C K A T G Q V C H A LC S P E G C W G P E P R D C V S V E C P P C P AP P V A G P S V F L F P P K P K D T L M I S R TP E V T C V V V D V S H E D P E V Q F N W Y V DG M E V H N A K T K P R E E Q F N S T F R V V SV L T V V H Q D W L N G K E Y K C K V S N K G LP A P I E K T I S K T K G Q P R E P Q V Y T L PP S R E E M T K N Q V S L T C L V K G F Y P S DI A V E W E S N G Q P E N N Y K T T P P M L D SD G S F F L Y S K L T V D K S R W Q Q G N V F SC S V M H E A L H N H Y T Q K S L S L S P G K Stop

EXAMPLE 10

The present example discloses a chimera containing a signal peptide (M EW S W V F L F F L S V T T G V H S) joined to the LI subdomain of theErbB4 receptor and the SI subdomain, the LII subdomain and module 1 ofthe SII subdomain of the ErbB1 receptor fused to IgG2Fc. The ErbBchimera has the following amino acid sequence:

SEQ. ID. NO. 10 M E W S W V F L F F L S V T T G V H S Q S V C AG T E N K L S S L S D L E Q Q Y R A L R K Y Y EN C E V V M G N L E I T S I E H N R D L S F L RS V R E V T G Y V L V A L N Q F R Y L P L E N LR I I R G T K L Y E D R Y A L A I F L N Y R K DG N F G L Q E L G L K N L T E I L N G G V Y V DQ N K F L C Y A D T I H W Q D I V R N P W P S NL T L V S T N G S S G C G R C D P S C P N G S CW G A G E E N C Q K L T K I I C A Q Q C S G R CR G K S P S D C C H N Q C A A G C T G P R E S DC L V C R K F R D E A T C K D T C P P L Y N L Y N P T T Y Q M D V N P E G K Y S F G A T C V K K C P R N Y V V T D H G S C V R A C G A D S Y E M E E D G V R K C K K C E G P C R K V C N G I G I G E F K D S L S I N A T N I K H F K N C T S I S G D L H I L P V A F R G D S F T H T P P L D P Q E L D I L K T V K E I T G F L L I Q A W P E N R T D L H A F E N L E I I R G R T K Q H G Q F S L A V V S L N I T S L G L R S L K E I S D G D V I I S G N K N L C Y A N T I N W K K L F G T S G Q K T K I I S N R G E N S C K A T G Q V C H A L C S P E G C W G P E P R D C V S V E C P P C P A P P V A G P S V F L F P P K P K D T L M I S R T P E V T C V V V D V S H E D P E V Q F N W Y V D G M E V H N A K T K P R E E Q F N S T F R V V S V L T V V H Q D W L N G K E Y K C K V S N K G L P A P I E K T I S K T K G Q P R E P Q V Y T L P P S R E E M T K N Q V S L T C L V K G F Y P S D I A V E W E S N G Q P E N N Y K T T P P M L D S D G S F F L Y S K L T V D K S R W Q Q G N V F S C S V M H E A L H N H Y T Q K S L S L S P G K Stop

EXAMPLE 11

The present example discloses a chimera containing a signal peptide (M EW S W V F L F F L S V T T G V H S) joined to the LI subdomain and the SIsubdomain of the ErbB4 receptor and LII subdomain and module 1 of the511 subdomain of the ErbB I receptor fused to IgG2Fe. The ErbB chimerahas the following amino acid sequence:

SEQ. ID. NO. 11 M E W S W V F L F F L S V T T G V H S Q S V C AG T E N K L S S L S D L E Q Q Y R A L R K Y Y EN C E V V M G N L E I T S I E H N R D L S F L RS V R E V T G Y V L V A L N Q F R Y L P L E N LR I I R G T K L Y E D R Y A L A I F L N Y R K DG N F G L Q E L G L K N L T E I L N G G V Y V DQ N K F L C Y A D T I H W Q D I V R N P W P S NL T L V S T N G S S G C G R C H K S C T G R C WG P T E N H C Q T L T R T V C A E Q C D G R C YG P Y V S D C C H R E C A G G C S G P K D T D CF A C M N F N D S G A C V T Q C P Q T F V Y N PT T F Q L E H N F N A K Y T Y G A F C V K K C PH N F V V D S S S C V R A C P S S K M E V E E NG I K M C K P C T D I C P K V C N G I G I G E FK D S L S I N A T N I K H F K N C T S I S G D LH I L P V A F R G D S F T H T P P L D P Q E L DI L K T V K E I T G F L L I Q A W P E N R T D LH A F E N L E I I R G R T K Q H G Q F S L A V VS L N I T S L G L R S L K E I S D G D V I I S GN K N L C Y A N T I N W K K L F G T S G Q K T KI I S N R G E N S C K A T G Q V C H A L C S P EG C W G P E P R D C V S V E C P P C P A P P V AG P S V F L F P P K P K D T L M I S R T P E V TC V V V D V S H E D P E V Q F N W Y V D G M E VH N A K T K P R E E Q F N S T F R V V S V L T VV H Q D W L N G K E Y K C K V S N K G L P A P IE K T I S K T K G Q P R E P Q V Y T L P P S R EE M T K N Q V S L T C L V K G F Y P S D I A V EW E S N G Q P E N N Y K T T P P M L D S D G S FF L Y S K L T V D K S R W Q Q G N V F S C S V MH E A L H N H Y T Q K S L S L S P G K Stop

EXAMPLE 12

The present example refers to a chimera containing a signal peptide (M EW S W V F L F F L S V T T G V H S) joined to the LI subdomain and themodules 1-2 of the SI subdomain of the ErbB4 receptor and the modules3-8 of the SI subdomain, the LII subdomain and module 1 of the SIIsubdomain of the ErbB1 receptor fused to IgG2Fc. The ErbB chimera hasthe following amino acid sequence:

SEQ. ID. NO. 12 M E W S W V F L F F L S V T T G V H S Q S V C AG T E N K L S S L S D L E Q Q Y R A L R K Y Y EN C E V V M G N L E I T S I E H N R D L S F L RS V R E V T G Y V L V A L N Q F R Y L P L E N LR I I R G T K L Y E D R Y A L A I F L N Y R K DG N F G L Q E L G L K N L T E I L N G G V Y V DQ N K F L C Y A D T I H W Q D I V R N P W P S NL T L V S T N G S S G C G R C H K S C T G R C WG P T E N H C Q T L T R T V C A E Q C D G R C YG P Y V S D C C H N Q C A A G C T G P R E S D CL V C R K F R D E A T C K D T C P P L M L Y N PT T Y Q M D V N P E G K Y S F G A T C V K K C PR N Y V V T D H G S C V R A C G A D S Y E M E ED G V R K C K K C E G P C R K V C N G I G I G EF K D S L S I N A T N I K H F K N C T S I S G DL H I L P V A F R G D S F T H T P P L D P Q E LD I L K T V K E I T G F L L I Q A W P E N R T DL H A F E N L E I I R G R T K Q H G Q F S L A VV S L N I T S L G L R S L K E I S D G D V I I SG N K N L C Y A N T I N W K K L F G T S G Q K TK I I S N R G E N S C K A T G Q V C H A L C S PE G C W G P E P R D C V S V E C P P C P A P P VA G P S V F L F P P K P K D T L M I S R T P E VT C V V V D V S H E D P E V Q F N W Y V D G M EV H N A K T K P R E E Q F N S T F R V V S V L TV V H Q D W L N G K E Y K C K V S N K G L P A PI E K T I S K T K G Q P R E P Q V Y T L P P S RE E M T K N Q V S L T C L V K G F Y P S D I A VE W E S N G Q P E N N Y K T T P P M L D S D G SF F L Y S K L T V D K S R W Q Q G N V F S C S VM H E A L H N H Y T Q K S L S L S P G K Stop

EXAMPLE 13

The present example refers to a chimera containing a signal peptide (M EW S W V F L F F L S V T T G V H S) joined to the LI subdomain and themodules 1-3 of the SI subdomain of the ErbB4 receptor and the modules4-8 of the SI subdomain, the LH subdomain and module 1 of the SIIsubdomain of the ErbB1 receptor fused to IgG2Fc. The ErbB chimera hasthe following amino acid sequence:

SEQ. ID. NO. 13 M E W S W V F L F F L S V T T G V H S Q S V C AG T E N K L S S L S D L E Q Q Y R A L R K Y Y EN C E V V M G N L E I T S I E H N R D L S F L RS V R E V T G Y V L V A L N Q F R Y L P L E N LR I I R G T K L Y E D R Y A L A I F L N Y R K DG N F G L Q E L G L K N L T E I L N G G V Y V DQ N K F L C Y A D T I H W Q D I V R N P W P S NL T L V S T N G S S G C G R C H K S C T G R C WG P T E N H C Q T L T R T V C A E Q C D G R C YG P Y V S D C C H R E C A G G C S G P K D T D CF V C R K F R D E A T C K D T C P P L M L Y N PT T Y Q M D V N P E G K Y S F G A T C V K K C PR N Y V V T D H G S C V R A C G A D S Y E M E ED G V R K C K K C E G P C R K V C N G I G I G EF K D S L S I N A T N I K H F K N C T S I S G DL H I L P V A F R G D S F T H T P P L D P Q E LD I L K T V K E I T G F L L I Q A W P E N R T DL H A F E N L E I I R G R T K Q H G Q F S L A VV S L N I T S L G L R S L K E I S D G D V I I SG N K N L C Y A N T I N W K K L F G T S G Q K TK I I S N R G E N S C K A T G Q V C H A L C S PE G C W G P E P R D C V S V E C P P C P A P P VA G P S V F L F P P K P K D T L M I S R T P E VT C V V V D V S H E D P E V Q F N W Y V D G M EV H N A K T K P R E E Q F N S T F R V V S V L TV V H Q D W L N G K E Y K C K V S N K G L P A PI E K T I S K T K G Q P R E P Q V Y T L P P S RE E M T K N Q V S L T C L V K G F Y P S D I A VE W E S N G Q P E N N Y K T T P P M L D S D G SF F L Y S K L T V D K S R W Q Q G N V F S C S VM H E A L H N H Y T Q K S L S L S P G K Stop

EXAMPLE 14

The present example discloses a chimera containing a signal peptide (M EW S W V F L F F L S V T T G V H S) joined to the LI subdomain and themodules 1-4 of the SI subdomain of the ErbB4 receptor and the modules5-8 of the SI subdomain, the LII subdomain and module 1 of the 511subdomain of the ErbB1 receptor fused to IgG2Fe. The ErbB chimera hasthe following amino acid sequence:

SEQ. ID. NO. 14 M E W S W V F L F F L S V T T G V H S Q S V C AG T E N K L S S L S D L E Q Q Y R A L R K Y Y EN C E V V M G N L E I T S I E H N R D L S F L RS V R E V T G Y V L V A L N Q F R Y L P L E N LR I I R G T K L Y E D R Y A L A I F L N Y R K DG N F G L Q E L G L K N L T E I L N G G V Y V DQ N K F L C Y A D T I H W Q D I V R N P W P S NL T L V S T N G S S G C G R C H K S C T G R C WG P T E N H C Q T L T R T V C A E Q C D G R C YG P Y V S D C C H R E C A G G C S G P K D T D CF A C M N F N D S G A C V T T C P P L M L Y N PT T Y Q M D V N P E G K Y S F G A T C V K K C PR N Y V V T D H G S C V R A C G A D S Y E M E ED G V R K C K K C E G P C R K V C N G I G I G EF K D S L S J N A T N I K H F K N C T S I S G DL H I L P V A F R G D S F T H T P P L D P Q E LD I L K T V K E I T G F L L I Q A W P E N R T DL H A F E N L E I I R G R T K Q H G Q F S L A VV S L N I T S L G L R S L K E I S D G D V I I SG N K N L C Y A N T I N W K K L F G T S G Q K TK I I S N R G E N S C K A T G Q V C H A L C S PE G C W G P E P R D C V S V E C P P C P A P P VA G P S V F L F P P K P K D T L M I S R T P E VT C V V V D V S H E D P E V Q F N W Y V D G M EV H N A K T K P R E E Q F N S T F R V V S V L TV V H Q D W L N G K E Y K C K V S N K G L P A PI E K T I S K T K G Q P R E P Q V Y T L P P S RE E M T K N Q V S L T C L V K G F Y P S D I A VE W E S N G Q P E N N Y K T T P P M L D S D G SF F L Y S K L T V D K S R W Q Q G N V F S C S VM H E A L H N H Y T Q K S L S L S P G K Stop

EXAMPLE 15

The present example discloses a chimera containing a signal peptide (M EW S W V F L F F L S V T T G V H S) joined to the LI subdomain and themodules 1-5 of the SI subdomain of the ErbB4 receptor and the modules6-8 of the SI subdomain, the LII subdomain and module 1 of the SIIsubdomain of the ErbB1 receptor fused to IgG2Fc. The ErbB chimera hasthe following amino acid sequence:

SEQ. ID. NO. 15 M E W S W V F L F F L S V T T G V H S Q S V C AG T E N K L S S L S D L E Q Q Y R A L R K Y Y EN C E V V M G N L E I T S I E H N R D L S F L RS V R E V T G Y V L V A L N Q F R Y L P L E N LR I I R G T K L Y E D R Y A L A I F L N Y R K DG N F G L Q E L G L K N L T E I L N G G V Y V DQ N K F L C Y A D T I H W Q D I V R N P W P S NL T L V S T N G S S G C G R C H K S C T G R C WG P T E N H C Q T L T R T V C A E Q C D G R C YG P Y V S D C C H R E C A G G C S G P K D T D CF A C M N F N D S G A C V T Q C P Q T F V Y N PT T F Q L E H N F N A K Y T Y G A F C V K K C PR N Y V V T D H G S C V R A C G A D S Y E M E ED G V R K C K K C E G P C R K V C N G I G I G EF K D S L S I N A T N I K H F K N C T S I S G DL H I L P V A F R G D S F T H T P P L D P Q E LD I L K T V K E I T G F L L I Q A W P E N R T DL H A F E N L E I I R G R T K Q H G Q F S L A VV S L N I T S L G L R S L K E I S D G D V I I SG N K N L C Y A N T I N W K K L F G T S G Q K TK I I S N R G E N S C K A T G Q V C H A L C S PE G C W G P E P R D C V S V E C P P C P A P P VA G P S V F L F P P K P K D T L M I S R T P E VT C V V V D V S H E D P E V Q F N W Y V D G M EV H N A K T K P R E E Q F N S T F R V V S V L TV V H Q D W L N G K E Y K C K V S N K G L P A PI E K T I S K T K G Q P R E P Q V Y T L P P S RE E M T K N Q V S L T C L V K G F Y P S D I A VE W E S N G Q P E N N Y K T T P P M L D S D G SF F L Y S K L T V D K S R W Q Q G N V F S C S VM H E A L H N H Y T Q K S L S L S P G K Stop

EXAMPLE 16

The present example refers to a chimera containing a signal peptide (M EW S W V F L F F L S V T T G V H S) joined to the LI subdomain and themodules 1-6 of the SI subdomain of the ErbB4 receptor and the modules7-8 of the SI subdomain, the LII subdomain and module 1 of the SIIsubdomain of the ErbB1 receptor fused to IgG2Fc. The ErbB chimera hasthe following amino acid sequence:

SEQ. ID. NO. 16 M E W S W V F L F F L S V T T G V H S Q S V C AG T E N K L S S L S D L E Q Q Y R A L R K Y Y EN C E V V M G N L E I T S I E H N R D L S F L RS V R E V T G Y V L V A L N Q F R Y L P L E N LR I I R G T K L Y E D R Y A L A I F L N Y R K DG N F G L Q E L G L K N L T E I L N G G V Y V DQ N K F L C Y A D T I H W Q D I V R N P W P S NL T L V S T N G S S G C G R C H K S C T G R C WG P T E N H C Q T L T R T V C A E Q C D G R C YG P Y V S D C C H R E C A G G C S G P K D T D CF A C M N F N D S G A C V T Q C P Q T F V Y N PT T F Q L E H N F N A K Y T Y G A F C V K K C PH N F V V D S S S C V R A C G A D S Y E M E E DG V R K C K K C E G P C R K V C N G I G I G E FK D S L S I N A T N I K H F K N C T S I S G D LH I L P V A F R G D S F T H T P P L D P Q E L DI L K T V K E I T G F L L I Q A W P E N R I D LH A F E N L E I I R G R T K Q H G Q F S L A V VS L N I T S L G L R S L K E I S D G D V I I S GN K N L C Y A N T I N W K K L F G T S G Q K T KI I S N R G E N S C K A T G Q V C H A L C S P EG C W G P E P R D C V S V E C P P C P A P P V AG P S V F L F P P K P K D T L M I S R T P E V TC V V V D V S H E D P E V Q F N W Y V D G M E VH N A K T K P R E E Q F N S T F R V V S V L T VV H Q D W L N G K E Y K C K V S N K G L P A P IE K T I S K T K G Q P R E P Q V Y T L P P S R EE M T K N Q V S L T C L V K G F Y P S D I A V EW E S N G Q P E N N Y K T T P P M L D S D G S FF L Y S K L T V D K S R W Q Q G N V F S C S V MH E A L H N H Y T Q K S L S L S P G K Stop

EXAMPLE 17

The present example refers to a chimera containing a signal peptide (M EW S W V F L F F L S V T T G V H S) joined to the LI subdomain and themodules 1-7 of the SI subdomain of the ErbB4 receptor and the module 8of the SI subdomain, the LII subdomain and module 1 of the SII subdomainof the ErbB1 receptor fused to IgG2Fc. The ErbB chimera has thefollowing amino acid sequence:

SEQ. ID. NO. 17 M E W S W V F L F F L S V T T G V H S Q S V C AG T E N K L S S L S D L E Q Q Y R A L R K Y Y EN C E V V M G N L E I T S I E H N R D L S F L RS V R E V T G Y V L V A L N Q F R Y L P L E N LR I I R G T K L Y E D R Y A L A I F L N Y R K DG N F G L Q E L G L K N L T E I L N G G V Y V DQ N K F L C Y A D T I H W Q D I V R N P W P S NL T L V S T N G S S G C G R C H K S C T G R C WG P T E N H C Q T L T R T V C A E Q C D G R C YG P Y V S D C C H R E C A G G C S G P K D T D CF A C M N F N D S G A C V T Q C P Q T F V Y N PT T F Q L E F I N F N A K Y T Y G A F C V K K CP H N F V V D S S S C V R A C P S S K M E V E EN G I K M C K P C E G P C R K V C N G I G I G EF K D S L S I N A T N I K H F K N C T S I S G DL H I L P V A F R G D S F T H T P P L D P Q E LD I L K T V K E I T G F L L I Q A W P E N R T DL H A F E N L E I I R G R T K Q H G Q F S L A VV S L N I T S L G L R S L K E I S D G D V I I SG N K N L C Y A N T I N W K K L F G T S G Q K TK I I S N R G E N S C K A T G Q V C H A L C S PE G C W G P E P R D C V S V E C P P C P A P P VA G P S V F L F P P K P K D T L M I S R T P E VT C V V V D V S H E D P E V Q F N W Y V D G M EV H N A K T K P R E E Q F N S T F R V V S V L TV V H Q D W L N G K E Y K C K V S N K G L P A PI E K T I S K T K G Q P R E P Q V Y T L P P S RE E M T K N Q V S L T C L V K G F Y P S D I A VE W E S N G Q P E N N Y K T T P P M L D S D G SF F L Y S K L T V D K S R W Q Q G N V F S C S VM H E A L H N H Y T Q K S L S L S P G K Stop

EXAMPLE 18

The present example discloses a chimera containing a signal peptide (M EW S W V F L F F L S V T T G V H S) joined to the LI subdomain and module1 of the SI subdomain of the ErbB4 receptor and modules 2-8 of the SIsubdomain, the LII subdomain and module 1 of the SII subdomain of theErbB1 receptor fused to IgG2Fc. The chimera has the following amino acidsequence:

SEQ. ID. NO. 18 M E W S W V F L F F L S V T T G V H S Q S V C AG T E N K L S S L S D L E Q Q Y R A L R K Y Y EN C E V V M G N L E I T S I E H N R D L S F L RS V R E V T G Y V L V A L N Q F R Y L P L E N LR I I R G T K L Y E D R Y A L A I F L N Y R K DG N F G L Q E L G L K N L T E I L N G G V Y V DQ N K F L C Y A D T I H W Q D I V R N P W P S NL T L V S T N G S S G C G R C H K S C T G R C WG P T E N H C Q T L T K I I C A Q Q C S G R C RG K S P S D C C H N Q C A A G C T G P R E S D CL V C R K F R D E A T C K D T C P P L M L Y N PT T Y Q M D V N P E G K Y S F G A T C V K K C PR N Y V V T D H G S C V R A C G A D S Y E M E ED G V R K C K K C E G P C R K V C N G I G I G EF K D S L S I N A T N I K H F K N C T S I S G DL H I L P V A F R G D S F T H T P P L D P Q E LD I L K T V K E I T G F L L I Q A W P E N R T DL H A F E N L E I I R G R T K Q H G Q F S L A VV S L N I T S L G L R S L K E I S D G D V I I SG N K N L C Y A N T I N W K K L F G T S G Q K TK H S N R G E N S C K A T G Q V C H A L C S P EG C W G P E P R D C V S V E C P P C P A P P V AG P S V F L F P P K P K D T L M I S R T P E V TC V V V D V S H E D P E V Q F N W Y V D G M E VH N A K T K P R E E Q F N S T F R V V S V L T VV H Q D W L N G K E Y K C K V S N K G L P A P IE K T I S K T K G Q P R E P Q V Y T L P P S R EE M T K N Q V S L T C L V K G F Y P S D I A V EW E S N G Q P E N N Y K T T P P M L D S D G S FF L Y S K L T V D K S R W Q Q G N V F S C S V M H E A L H N H Y T Q K S L S L S P G K Stop

EXAMPLE 19

The present example discloses a chimera containing a signal peptide (M EW S W V F L F F L S V T T G V H S) joined to the LI subdomain andmodules 1-5 of the SI subdomain of the ErbB4 receptor (through aminoacid residue 245 of the ErbB4 receptor in the alignment of FIG. 3) andmodules 5-8 of the SI subdomain of the ErbB1 receptor (starting at aminoacid residue 249 of the ErbB1 receptor in the alignment of FIG. 3), theLII subdomain and module 1 of the SII subdomain of the ErbB1 receptor,fused to IgG2Fc after amino acid 501 in the SII subdomain. Amino acid245 is based on numbering of the ErbB4 receptor while amino acids 249and 501 are based on numbering of the ErbB1 receptor. The chimera hasthe following amino acid sequence:

SEQ. ID. NO. 19 M E W S W V F L F F L S V T T G V H S Q S V C AG T E N K L S S L S D L E Q Q Y R A L R K Y Y EN C E V V M G N L E I T S I E H N R D L S F L RS V R E V T G Y V L V A L N Q F R Y L P L E N LR I I R G T K L Y E D R Y A L A I F L N Y R K DG N F G L Q E L G L K N L T E I L N G G V Y V DQ N K F L C Y A D T I H W Q D I V R N P W P S NL T L V S T N G S S G C G R C H K S C T G R C WG P T E N H C Q T L T R T V C A E Q C D G R C YG P Y V S D C C H R E C A G G C S G P K D T D CF A C M N F N D S G A C V T Q C P Q T F V Y N PT T Y Q M D V N P E G K Y S F G A T C V K K C PR N Y V V T D H G S C V R A C G A D S Y E M E ED G V R K C K K C E G P C R K V C N G I G I G EF K D S L S I N A T N I K H F K N C T S I S G DL H I L P V A F R G D S F T H T P P L D P Q E LD I L K T V K E I T G F L L I Q A W P E N R T DL H A F E N L E I I R G R T K Q H G Q F S L A VV S L N I T S L G L R S L K E I S D G D V I I SG N K N L C Y A N T I N W K K L F G T S G Q K TK I I S N R G E N S C K A T G Q V C H A L C S PE G C W G P E P R D C V S V E C P P C P A P P VA G P S V F L F P P K P K D T L M I S R T P E VT C V V V D V S H E D P E V Q F N W Y V D G M EV H N A K T K P R E E Q F N S T F R V V S V L TV V H Q D W L N G K E Y K C K V S N K G L P A PI E K T I S K T K G Q P R E P Q V Y T L P P S RE E M T K N Q V S L T C L V K G F Y P S D I A VE W E S N G Q P E N N Y K T T P P M L D S D G SF F L Y S K L T V D K S R W Q Q G N V F S C S VM H E A L H N H Y T Q K S L S L S P G K Stop

EXAMPLE 20

The present example discloses a chimera containing the LI subdomain andmodules 1-5 of the SI subdomain of the ErbB4 receptor (through aminoacid residue 245 of the ErbB4 receptor in the alignment of FIG. 3) andmodules 5-8 of the SI subdomain of the ErbB1 receptor (starting at aminoacid residue 249 of the ErbB1 receptor in the alignment of FIG. 3), theLII subdomain and module 1 of the S11 subdomain of the ErbB1 receptor,fused to IgG2Fc after amino acid 501 in the SII subdomain. Amino acid245 is based on numbering of the ErbB4 receptor while amino acids 249and 501 are based on numbering of the ErbB1 receptor. Additionally, thissequence contains 2 cysteine (Cys) to serine (Ser) substitutions in thehinge region of IgG2Fc. These substitutions are C226S and C229S, basedon numbering of the IgG2Fc protein and are underlined below. The chimerahas the following amino acid sequence:

SEQ. ID. NO. 20 M E W S W V F L F F L S V T T G V H S Q S V C AG T E N K L S S L S D L E Q Q Y R A L R K Y Y EN C E V V M G N L E I T S I E H N R D L S F L RS V R E V T G Y V L V A L N Q F R Y L P L E N LR I I R G T K L Y E D R Y A L A I F L N Y R K DG N F G L Q E L G L K N L T E I L N G G V Y V DQ N K F L C Y A D T I H W Q D I V R N P W P S NL T L V S T N G S S G C G R C H K S C T G R C WG P T E N H C Q T L T R T V C A E Q C D G R C YG P Y V S D C C H R E C A G G C S G P K D T D CF A C M N F N D S G A C V T Q C P Q T F V Y N PT T Y Q M D V N P E G K Y S F G A T C V K K C PR N Y V V T D H G S C V R A C G A D S Y E M E ED G V R K C K K C E G P C R K V C N G I G I G EF K D S L S I N A T N I K H F K N C T S I S G DL H I L P V A F R G D S F T H T P P L D P Q E LD I L K T V K E I T G F L L I Q A W P E N R T DL H A F E N L E I I R G R T K Q H G Q F S L A VV S L N I T S L G L R S L K E I S D G D V I I SG N K N L C Y A N T I N W K K L F G T S G Q K TK I I S N R G E N S C K A T G Q V C H A L C S PE G C W G P E P R D C V S V E S P P S P A P P VA G P S V F L F P P K P K D T L M I S R T P E VT C V V V D V S H E D P E V Q F N W Y V D G M EV H N A K T K P R E E Q F N S T F R V V S V L TV V H Q D W L N G K E Y K C K V S N K G L P A PI E K T I S K T K G Q P R E P Q V Y T L P P S RE E M T K N Q V S L T C L V K G F Y P S D I A VE W E S N G Q P E N N Y K T T P P M L D S D G SF F L Y S K L T V D K S R W Q Q G N V F S C S VM H E A L H N H Y T Q K S L S L S P G K Stop

EXAMPLE 21

This example demonstrates that ErbB chimeras having substantial bindingaffinity for ligands to both receptors can be created from the selectivecombination of two different ErbB receptors. Four constructs were testedfor binding to HRG1β and TGFα including the construct from Example 19(Seq ID No. 19), designated for purposes of this example as E1/E4-Fc,and the construct from Example 20 (Seq ID No. 20) which is designatedE1/E4-MFc. The controls included amino acids 1-501 of ErbB1 joined tothe IgG2Fc portion, designated E1 and amino acids 1-497 of ErbB4 joinedto the IgG2Fc portion, designated E4. In some studies another E1 controlwas used.

Trap DNA molecules were synthesized by starting with the desired aminoacid sequence and optimizing the DNA sequence for mammalian systemexpression. Trap DNA sequences were cloned into a suitable mammalianexpression vector (pCpGfree-vitroHmcs) that can be selected usinghygromycin and contains MAR/SAR sequences (insulator and boundaryregions) and promoters and enhancers for trap expression. Vectorscontaining trap sequences were transfected into CHO cells by standardtransfection methods and the cells were selected for vector integrationwith hygromycin. Traps were purified from stably transfected cell linesby collecting cell culture medium and purifying by standard methods(protein A column binding). Traps were eluted from protein A by standardmethods and quantitated using a custom derived IgG-Fc sandwich ELISAassay.

Traps were purified using protein A and run on a polyacrylamide gel,under non-reducing (NR) and reducing (R) conditions. Disulfide linkeddimers run at approximately 220-240 kDa, while reduced monomers run at120-130 kDa. The chimeric trap monomer with mutated cysteines thatprevent disulfide formation runs at approximately 120-130 kDa under bothnon-reducing and reducing conditions. The E1-Fc, E4-Fc, E1/E4-Fcappeared to be in the 220-240 kDa range when run under nonreducingconditions and in the range of about 120-130 when run under reducingconditions. The E1/E4-Fc construct appeared to have molecular weight ofabout 120-130 under both nonreducing and reducing conditions.

Purified trap molecules were coated on 96 well plates and incubated witheither TGFα or HRG1β. Then detection antibodies against TGFα or HRG1βwere used to measure the affinity (Kd) and amount of bound ligand(Bmax).

Traps were coated on 96 well plates and incubated with varyingconcentrations of either TGFα or HRG1β. Detection antibodies againstTGFα or HRG1β were used to measure the affinity (Kd) and amount of boundligand (Bmax). Data obtained in these studies is provided in Tables 1-4below.

As set forth in Tables 1 and 2, the chimeric monomer (E1/E4-Fc) boundTGFα with an affinity in the range of about 400-620 nM and ErbB1-IgGFc(E1-Fc) bound with an affinity (Kd) in the range of about 27-40 nM.

TABLE 1 TGFα Binding Study E1-Fc E4-Fc E1/E4-Fc E1/E4-MFc E1-controlBmax 970,300 1,249,000 2,410,000 2,509,000 1,118,000 Kd (nM) 27.48 4.895676.7 406.5 18.47 Std Error Bmax 101,600 24,510 478,600 392,300 73,470Std Error 12.69 5.887 266.2 149 5.72 Kd (nM)

TABLE 2 TGFα Binding Study E1/E4- E1/E4M- E1-Fc E4-Fc E1/E4-Fc MFc 50%Bmax 781,267 280,121 834,302 695,464 779,310 Kd (nM) 40.33 10601 344.8621.2 1953 Std Error 45,287 2,180,000 113,865 137,557 338,644 Bmax StdError 9.847 89561 116.3 250.5 1208 Kd (nM)

As shown in Tables 3 and 4, the chimeric monomer (E1/E4-Fc) bound HRG1βwith an affinity in the range of about approximately 15-30 nM which wasessentially the same as ErbB4-IgGFc (E4-Fc).

TABLE 3 HRG1β Binding Study E1/E4-Fc E1/E4-Fc BATCH1 BATCH 2 E1/E4-MFcE4-Fc Bmax 1,460,000 1,630,000 4,440,000 2,880,000 Kd 13.1 17.3 27.7721.18 (nM) Std 38,008 56,194 176,175 120,882 Error Bmax Std 1.255 2.0553.375 2.92 Error Kd (nM)

TABLE 4 HRG1β Binding Study E1/E4-MFc E1/E4-Fc E1/E4-MFc 50% E4-Fc Bmax1,900,000 6,360,000 3,110,000 1,480,000 Kd 23.82 26.62 15.46 14.51 (nM)Std 135,806 876,498 179,457 117,757 Error Bmax Std 3.932 8.175 2.3663.128 Error Kd (nM)

The E1/E4-MFc 50% data point shows that when one half of the amount ofthe purified chimeric receptor is added to a well that binding goes downby half and therefore binding is proportional to the amount of addedreceptor.

This example demonstrates that ErbB chimeras can be created and purifiedthat have substantial affinity for ligands to both of its receptorsubcomponents. Further this example shows that mutations can beintroduced into the chimeric receptor binding molecules to changedisulfides to prevent the formation of dimers through disulfide bondformation.

EXAMPLE 22

The present example discloses a chimera containing a signal peptide (M EW S W V F L F F L S V T T G V H S) joined to amino acids 1-245 of theErbB4 receptor and amino acids 249 to 501 of the ErbB1 receptor. Thefusion also includes IgG2-Fc as in the sequences above. Alsoincorporated into this sequence is a substitution of glutamine forserine at position 13 and the two cysteine to serine modifications fromexample 20 that prevent disulfide based dimerization of the molecule.The glutamine at position 13 (underlined below) is found in the ErbB1sequence at that location and is incorporated in the sequence toincrease the affinity of the chimera for TGFα. The ErbB chimera has thefollowing amino acid sequence:

SEQ. ID. NO. 21 M E W S W V F L F F L S V T T G V H S Q S V C AG T E N K L S Q L S D L E Q Q Y R A L R K Y Y EN C E V V M G N L E I T S I E H N R D L S F L RS V R E V T G Y V L V A L N Q F R Y L P L E N LR I I R G T K L Y E D R Y A L A I F L N Y R K DG N F G L Q E L G L K N L T E I L N G G V Y V DQ N K F L C Y A D T I H W Q D I V R N P W P S NL T L V S T N G S S G C G R C H K S C T G R C WG P T E N H C Q T L T R T V C A E Q C D G R C YG P Y V S D C C H R E C A G G C S G P K D T D CF A C M N F N D S G A C V T Q C P Q T F V Y N PT T Y Q M D V N P E G K Y S F G A T C V K K C PR N Y V V T D H G S C V R A C G A D S Y E M E ED G V R K C K K C E G P C R K V C N G I G I G EF K D S L S I N A T N I K H F K N C T S I S G DL H I L P V A F R G D S F T H T P P L D P Q E LD I L K T V K E I T G F L L I Q A W P E N R T DL H A F E N L E I I R G R T K Q H G Q F S L A VV S L N I T S L G L R S L K E I S D G D V I I SG N K N L C Y A N T I N W K K L F G T S G Q K TK I I S N R G E N S C K A T G Q V C H A L C S PE G C W G P E P R D C V S V E S P P S P A P P VA G P S V F L F P P K P K D T L M I S R T P E VT C V V V D V S H E D P E V Q F N W Y V D G M EV H N A K T K P R E E Q F N S T F R V V S V L TV V H Q D W L N G K E Y K C K V S N K G L P A PI E K T I S K T K G Q P R E P Q V Y T L P P S RE E M T K N Q V S L T C L V K G F Y P S D I A VE W E S N G Q P E N N Y K T T P P M L D S D G SF F L Y S K L T V D K S R W Q Q G N V F S C S VM H E A L H N H Y T Q K S L S L S P G K Stop

EXAMPLE 23

The present example discloses a chimera containing a signal peptide (M EW S W V F L F F L S V T T G V H S) joined to amino acids 1-245 of theErbB4 receptor and amino acids 249 to 501 of the ErbB1 receptor. Thefusion also includes IgG2-Fc as in the sequences above. Alsoincorporated into this sequence is a substitution of tyrosine for serineat position 42 and the two cysteine to serine modifications from example20 that prevent disulfide based dimerization of the molecule. Thetyrosine (underlined below) at position 42 is found in the ErbB1sequence at that location and is incorporated in the sequence toincrease the affinity of the chimera for TGFα. The ErbB chimera has thefollowing amino acid sequence:

SEQ. ID. NO. 22 M E W S W V F L F F L S V T T G V H S Q S V C AG T E N K L S S L S D L E Q Q Y R A L R K Y Y EN C E V V M G N L E I T Y I E H N R D L S F L RS V R E V T G Y V L V A L N Q F R Y L P L E N LR I I R G T K L Y E D R Y A L A I F L N Y R K DG N F G L Q E L G L K N L T E I L N G G V Y V DQ N K F L C Y A D T I H W Q D I V R N P W P S NL T L V S T N G S S G C G R C H K S C T G R C WG P T E N H C Q T L T R T V C A E Q C D G R C YG P Y V S D C C H R E C A G G C S G P K D T D CF A C M N F N D S G A C V T Q C P Q T F V Y N PT T Y Q M D V N P E G K Y S F G A T C V K K C PR N Y V V T D H G S C V R A C G A D S Y E M E ED G V R K C K K C E G P C R K V C N G I G I G EF K D S L S I N A T N I K H F K N C T S I S G DL H I L P V A F R G D S F T H T P P L D P Q E LD I L K T V K E I T G F L L I Q A W P E N R T DL H A F E N L E I I R G R T K Q H G Q F S L A VV S L N I T S L G L R S L K E I S D G D V I I SG N K N L C Y A N T I N W K K L F G T S G Q K TK I I S N R G E N S C K A T G Q V C H A L C S PE G C W G P E P R D C V S V E S P P S P A P P VA G P S V F L F P P K P K D T L M I S R T P E VT C V V V D V S H E D P E V Q F N W Y V D G M EV H N A K T K P R E E Q F N S T F R V V S V L TV V H Q D W L N G K E Y K C K V S N K G L P A PI E K T I S K T K G Q P R E P Q V Y T L P P S RE E M T K N Q V S L T C L V K G F Y P S D I A VE W E S N G Q P E N N Y K T T P P M L D S D G SF F L Y S K L T V D K S R W Q Q G N V F S C S VM H E A L H N H Y T Q K S L S L S P G K Stop

EXAMPLE 24

The present example discloses a chimera containing a signal peptide (M EW S W V F L F F L S V T T G V H S) joined to amino acids 1-245 of theErbB4 receptor and amino acids 249 to 501 of the ErbB1 receptor. Thefusion also includes IgG2-Fc as in the sequences above. Alsoincorporated into this sequence is a substitution of arginine fortyrosine at position 123 (underlined) and the two cysteine to serinemodifications from example 20 that prevent disulfide based dimerizationof the molecule. The arginine at position 123 is found in the ErbB1sequence at that location and is incorporated in the sequence toincrease the affinity of the chimera for TGFα. The ErbB chimera has thefollowing amino acid sequence:

SEQ. ID. NO. 23 M E W S W V F L F F L S V T T G V H S Q S V C AG T E N K L S S L S D L E Q Q Y R A L R K Y Y EN C E V V M G N L E I T S I E H N R D L S F L RS V R E V T G Y V L V A L N Q F R Y L P L E N LR I I R G T K L Y E D R Y A L A I F L N Y R K DG N F G L Q E L G L K N L T E I L N G G V R V DQ N K F L C Y A D T I H W Q D I V R N P W P S NL T L V S T N G S S G C G R C H K S C T G R C WG P T E N H C Q T L T R T V C A E Q C D G R C YG P Y V S D C C H R E C A G G C S G P K D T D CF A C M N F N D S G A C V T Q C P Q T F V Y N PT T Y Q M D V N P E G K Y S F G A T C V K K C PR N Y V V T D H G S C V R A C G A D S Y E M E ED G V R K C K K C E G P C R K V C N G I G I G EF K D S L S I N A T N I K H F K N C T S I S G DL H I L P V A F R G D S F T H T P P L D P Q E LD I L K T V K E I T G F L L I Q A W P E N R T DL H A F E N L E I I R G R T K Q H G Q F S L A VV S L N I T S L G L R S L K E I S D G D V I I SG N K N L C Y A N T I N W K K L F G T S G Q K TK I I S N R G E N S C K A T G Q V C H A L C S PE G C W G P E P R D C V S V E S P P S P A P P VA G P S V F L F P P K P K D T L M I S R T P E VT C V V V D V S H E D P E V Q F N W Y V D G M EV H N A K T K P R E E Q F N S T F R V V S V L TV V H Q D W L N G K E Y K C K V S N K G L P A PI E K T I S K T K G Q P R E P Q V Y T L P P S RE E M T K N Q V S L T C L V K G F Y P S D I A VE W E S N G Q P E N N Y K T T P P M L D S D G SF F L Y S K L T V D K S R W Q Q G N V F S C S VM H E A L H N H Y T Q K S L S L S P G K Stop

EXAMPLE 25

The present example discloses a chimera containing a signal peptide (M EW S W V F L F F L S V T T G V H S) joined to amino acids 1-183 of theErbB4 receptor and amino acids 187-501 of the ErbB1 receptor. Thesequence is essentially the same as the sequence in Example 18, exceptthat it contains two cysteine to serine modifications in the IgG2Fcregion to prevent dimerization (as in example 20). This fusion containsmore ErbB1 sequence in order to increase the binding affinity of thechimera for TGFα. The ErbB chimera has the following amino acidsequence:

SEQ. ID. NO. 24 M E W S W V F L F F L S V T T G V H S Q S V C AG T E N K L S S L S D L E Q Q Y R A L R K Y Y EN C E V V M G N L E I T S I E H N R D L S F L RS V R E V T G Y V L V A L N Q F R Y L P L E N LR I I R G T K L Y E D R Y A L A I F L N Y R K DG N F G L Q E L G L K N L T E I L N G G V Y V DQ N K F L C Y A D T I H W Q D I V R N P W P S NL T L V S T N G S S G C G R C H K S C T G R C WG P T E N H C Q T L T K I I C A Q Q C S G R C RG K S P S D C C H N Q C A A G C T G P R E S D CL V C R K F R D E A T C K D T C P P L M U N P TT Y Q M D V N P E G K Y S F G A T C V K K C P RN Y V V T D H G S C V R A C G A D S Y E M E E DG V R K C K K C E G P C R K V C N G I G I G E FK D S L S I N A T N I K H F K N C T S I S G D LH I L P V A F R G D S F T H T P P L D P Q E L DI L K T V K E I T G F L L I Q A W P E N R T D LH A F E N L E I I R G R T K Q H G Q F S L A V VS L N I T S L G L R S L K E I S D G D V I I S GN K N L C Y A N T I N W K K L F G T S G Q K T KI I S N R G E N S C K A T G Q V C H A L C S P EG C W G P E P R D C V S V E S P P S P A P P V AG P S V F L F P P K P K D T L M I S R T P E V TC V V V D V S H E D P E V Q F N W Y V D G M E VH N A K T K P R E E Q F N S T F R V V S V L T VV H Q D W L N G K E Y K C K V S N K G L P A P IE K T I S K T K G Q P R E P Q V Y T L P P S R EE M T K N Q V S L T C L V K G F Y P S D I A V EW E S N G Q P E N N Y K T T P P M L D S D G S FF L Y S K L T V D K S R W Q Q G N V F S C S V MH E A L H N H Y T Q K S L S L S P G K Stop

The invention claimed is:
 1. A chimeric ErbB ligand binding moleculemonomer comprising, L1, S1, L2 domains from an ErbB receptor; wherein L1is from ErbB4, L2 is from ErbB1, and wherein S1 is comprised of m1, m2,m3, m4, m5, m6, m7, and m8 subdomains; wherein the m1, m2, m3, and m4subdomains are from ErbB4 and the m6, m7, and m8 subdomains are fromErbB1 and the m5 subdomain has a sequence that is derived from ErbB4 andErbB1, wherein the chimeric ErbB ligand binding molecule can be purifiedfrom collected cell culture medium.
 2. The chimeric ErbB ligand bindingmolecule monomer of claim 1 further comprising, an immunoglobulin Fcportion.
 3. A chimeric ErbB ligand binding molecule monomer comprising,L1, S1, L2 domains from an ErbB receptor; wherein L1 is from ErbB4, L2is from ErbB1, and wherein S1 is comprised of m1, m2, m3, m4, m5, m6,m7, and m8 subdomains; wherein the m1, m2, m3, and m4 subdomains arefrom ErbB4 and the m6, m7, and m8 subdomains are from ErbB1 and the m5subdomain has a sequence that is derived from ErbB4 and ErbB1, whereinthe chimeric ErbB ligand binding molecule can be purified from collectedcell culture medium, wherein the chimeric ErbB ligand binding moleculeis fused to an immunoglobulin Fc portion and wherein the chimeric ErbBligand binding molecule/Fc fusion is dimerized.